Broadband absorbing film for laser capture microdissection

ABSTRACT

A thermoplastic film for LCM tissue transfer is thermally coupled to a broadband energy-absorbing material. The broadband energy-absorbing material may either be introduced into the film composition as a dopant or may be in thermal contact with the film.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 08/800,882, filed on Feb. 14, 1997, the contents of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to laser capture microdissection.More particularly, the present invention relates to broadband absorbingfilms for use in laser capture microdissection.

[0004] 2. The Prior Art

[0005] The LCM technique is generally described in the recentlypublished article: Laser Capture Microdissection, Science, Volume 274,Number 5289, Issue 8, pp 998-1001, published in 1996, incorporatedherein by reference. The purpose of the LCM technique is to provide asimple method for the procurement of selected human cells from aheterogeneous population contained on a typical histopathology biopsyslide.

[0006] A typical tissue biopsy sample consists of a 5 to 10 micron sliceof tissue that is placed on a glass microscope slide using techniqueswell known in the field of pathology. This tissue slice is a crosssection of the body organ that is being studied. The tissue consists ofa variety of different types of cells. Often a pathologist desires toremove only a small portion of the tissue for further analysis.

[0007] LCM employs a thermoplastic transfer film that is placed on topof the tissue sample. This film is manufactured containing organic dyesthat are chosen to selectively absorb in the near infrared region of thespectrum overlapping the emission region of common AlGaAs laser diodes.When the film is exposed to the focused laser beam the exposed region isheated by the laser and melts, adhering to the tissue in the region thatwas exposed. The film is then lifted from the tissue and the selectedportion of the tissue is removed with the film.

[0008] Thermoplastic transfer films such as a 100 micron thick ethylvinyl acetate (EVA) film available from Electroseal Corporation ofPompton Lakes, N.J. (type E540) have been used in LCM applications. Thefilm is chosen to have a low melting point of about 90 C.

[0009] The thermoplastic EVA films used in LCM techniques have beendoped with dyes, such as an infrared napthalocyanine dye, available fromAldrich Chemical Company (dye number 43296-2 or 39317-7). These dyeshave a strong absorption in the 800 nm region, a wavelength region thatoverlaps with laser emitters used to selectively melt the film. The dyeis mixed with the melted bulk plastic at an elevated temperature. Thedyed plastic is then manufactured into a film using standard filmmanufacturing techniques. The dye concentration in the plastic is about0.001M.

[0010] While the films employed in LCM applications have provedsatisfactory for the task, they have several drawbacks. The opticalabsorption of a dye impregnated film is a function of its thickness.This property of the film may be in conflict with a desire to selectfilm thickness for other reasons.

[0011] The organic dyes which are used to alter the absorptioncharacteristics of the films may have detrimental photochemistry effectsin some cases. This could result in contamination of LCM samples. Inaddition, the organic dyes employed to date are sensitive to thewavelength of the incident laser light and thus the film must be matchedto the laser employed.

[0012] It is therefore an object of the invention to provide a film forLCM applications, which overcomes the shortcomings of the prior art.

[0013] It is another object of the present invention to provide a simpleLCM film to be used in an LCM instrument.

[0014] Yet another object of the present invention is to provide an LCMfilm which is very thin and can have very high optical absorption.

[0015] A further object of the present invention is to provide anoptical quality LCM film, which is not sensitive to the wavelength ofthe incident laser light.

[0016] A further object of the present invention is to provide opticalquality LCM films of adjustable thickness.

[0017] Another object of the present invention is to provide an LCM filmwhose optical density can be adjusted so that sufficient light istransmitted through the sample to permit the sample to be viewed withback light illumination.

[0018] Yet another object of the present invention is to provide an LCMfilm, which does not use an organic dye that might have detrimentalphotochemistry effects.

[0019] Yet another object of the present invention is to provide an LCMfilm, which does not employ an organic dye, which does not have to bedissolved into the EVA polymer at high concentrations.

BRIEF SUMMARY OF THE INVENTION

[0020] A thermoplastic film for LCM tissue transfer is thermally coupledto a broadband energy-absorbing material. The broadband energy-absorbingmaterial may either be introduced into the film composition as a dopantor may be in thermal contact with the film.

[0021] According to one embodiment of the present invention, a film forLCM tissue transfer comprises a layer of a transparent support film; alayer of a broadband absorbing film; and a layer of a low temperaturethermoplastic; the absorbing film having an overall thickness chosen toabsorb a desired fraction of incident laser light.

[0022] According to this embodiment of the invention, the film consistsof a sandwich of layers containing a transparent support film such asmylar or polyester, a broadband absorbing film such as a metal film ofnichrome or titanium, and a low temperature thermoplastic layer such asethyl vinyl alcohol (EVA). The metal film thickness is chosen so as toabsorb a desired fraction of incident laser light. A typical film mayabsorb say 10% to 90% of the incident light. The metal thickness isadjusted to provide this optical density by controlling the length oftime of the sputtering deposition process. The metal absorbing layer canbe sandwiched between two layers of EVA if desired.

[0023] A method for fabricating a multilayer film for LCM tissuetransfer comprises the steps of: providing a support layer; forming athin layer of a broadband absorbing film onto the support layer, thebroadband absorbing film having a thickness chosen to absorb a desiredfraction of incident laser light; and coating the broadband absorbingfilm with a layer of thermoplastic.

[0024] According to the method of the present invention, one embodimentof the film is fabricated by evaporating a thin layer of metal film ontothe polyester support using a deposition technique such as sputtering.The film thickness is chosen so as to absorb a desired fraction ofincident laser light, usually between 10% and 95%. The metal thicknessis adjusted to provide this optical density by controlling the length oftime of the sputtering deposition process. The polyester/metal film isthen coated with a thin layer (a few to 100 microns) of EVAthermoplastic using a spin coater to achieve a uniform layer across thesurface. The spin coating process can be repeated several times toadjust the thickness of the EVA film.

[0025] According to one embodiment of the present invention, athermoplastic film is doped with a broadband energy-absorbing material.The dopant concentration is adjusted so as to give a suitableabsorption, usually between 10% and 95%, for the desired thickness ofEVA film.

[0026] According to another embodiment of the present invention, abroadband absorbing material is deposited on a substrate such as a capfor a biological analysis vessel. The metal film is deposited onto thebottom of a cap and then the cap is coated with EVA using the processthat is disclosed in the prior application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a cross sectional view of an LCM tissue transfer film ina multiplayer structure according to a first embodiment of the presentinvention.

[0028]FIG. 2 is a cross sectional view of an LCM tissue transfer film ina multilayer structure according to a second embodiment of the presentinvention.

[0029]FIG. 3 is a cross sectional view of an LCM tissue transfer filmaccording to a second embodiment of the present invention wherein abroadband energy-absorbing material is doped into the transfer film.

[0030]FIG. 4 is a cross sectional view of an LCM tissue transfer filmaccording to a second embodiment of the present invention wherein abroadband energy-absorbing material is formed on a substrate.

[0031]FIG. 5 is a perspective view of a cap for an analysis vesselshowing a procedure for affixing the LCM film thereto.

[0032]FIG. 6 is a perspective view of a cap for an analysis vesselshowing an alternate procedure for affixing the LCM film thereto.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Those of ordinary skill in the art will realize that thefollowing description of the present invention is illustrative only andnot in any way limiting. Other embodiments of the invention will readilysuggest themselves to such skilled persons.

[0034] Reference is made in the following disclosure to ethyl vinylacetate (EVA) film as the transfer film. Persons of ordinary skill inthe art will recognize that other films having desirable properties foruse as the transfer film may also be employed for that purpose and thedisclosure is not intended to be limited to LCM films employing EVAtransfer films.

[0035] The present invention comprises an LCM film employing a broadbandenergy-absorbing material either doped into the layer of transfer filmmaterial or as a film in thermal contact with the layer of transfer filmmaterial. The broadband absorber should have a relatively broadabsorption line throughout the visible region of the spectrum so thatthe absorber does not effect the color spectrum of the transmitted lightthat is used to illuminate the sample.

[0036] Metal films, such as nichrome or titanium are examples of abroadband absorber that can be deposited on a surface, which can bethermally coupled to the transfer film. Thermal coupling to the transferfilm may be accomplished by evaporating a thin layer of broadbandabsorbing film onto a transparent support film such as mylar orpolyester.

[0037] Referring first to FIG. 1, a cross sectional view of an LCMtissue transfer film 10 according to a first embodiment of the presentinvention is presented. The film 10 consists of a sandwich of layersincluding a transparent support film 12 such as mylar or polyester, abroadband absorbing film 14 such as a metal film of nichrome ortitanium, and a low temperature thermoplastic layer 16 such as EVA.

[0038] The transparent support layer may have a thickness of betweenabout 25 microns and about 250 microns. The thickness of the broadbandabsorbing film is chosen so as to absorb a desired fraction of incidentlaser light. A typical film may absorb from about 10% to about 95% ofthe incident light. The metal thickness is adjusted to provide thisoptical density by controlling the length of time of the sputteringdeposition process.

[0039] The film 10 of FIG. 1 is fabricated by evaporating a thin layerof broadband absorbing film onto the support film using a depositiontechnique such as sputtering as is known in the art. Another method fordepositing metal film coatings is to use electron beam evaporationmethods, which are well known in the art. The appropriate thickness ofthe metal film can be determined by monitoring the film depositionprocess using a witness sample that allows measurement of the opticaltransmission of the film as a function of deposition time. Thedeposition is halted when the appropriate transmission level is reached.This is a procedure that is well known to those skilled in the art ofthin film coating.

[0040] According to a presently preferred embodiment of the invention,the support and broadband absorbing film sandwich is then coated with athin layer (between a few microns and about 100 microns) of EVAthermoplastic using a spin coater to achieve a uniform layer across thepiece. The spin coating process can be repeated several times to adjustthe thickness of the EVA transfer film. If necessary, the EVA materialmay be dissolved in a solvent such as methylene chloride to reduce itsviscosity as is well known to those of ordinary skill in the art.

[0041] Referring now to FIG. 2, a cross sectional view of an LCM tissuetransfer film 20 according to a second embodiment of the presentinvention is presented. The film 20 consists of a sandwich of layersincluding a transparent support film 22 such as mylar or polyester, afirst low temperature thermoplastic layer 24 such as EVA, a broadbandabsorbing film 26 such as a metal film of nichrome or titanium, and asecond low temperature thermoplastic layer 28 such as EVA.

[0042] The process for making the film of FIG. 2 is similar to that usedto make the film of FIG. 1. The additional EVA layer may be formed by aspin-on process.

[0043] In use, the EVA side of the film of either FIGS. 1 or 2 is placedin contact with a thin tissue sample. Laser light is focused on thesandwich structure and the light is absorbed by the thin, metal film,which raises the temperature of the EVA film and melts the portion ofthe EVA that is in contact with the region of the metal film that isexposed to the focused laser beam. The laser power is chosen that thetemperature rise does not melt the polyester support film. For filmscontemplated by the present invention, typical laser powers are on theorder of 50 mW for film transmissions of about 10%. The melted EVA areaadheres to tissue samples, which can then be excised by removing thefilm.

[0044] Referring now to FIG. 3, a cross sectional view is presented ofan LCM tissue transfer film 30 according to a second embodiment of thepresent invention wherein a broadband energy-absorbing material is dopedinto the transfer film. The broadband energy-absorbing material isillustrated as shaded region 32.

[0045] An example of a broadband absorber that can be used to dope theEVA plastic is the chemical substance Buckminsterfullerene available asproduct #379646 from Sigma Chemical Company of St. Louis, Mo. Thissubstance is a pure carbon compound comprising submicron particles thathave very broad absorption profiles in the visible region of thespectrum. The Buckminsterfullerene can be mixed with heated EVA and theconcentration adjusted so as to give a suitable absorption, usuallybetween 10% and 95%, for the desired thickness of EVA film. Theadvantage of this method of doping the film is that, unlike the priorart LCM transfer films containing organic dyes, a specific laserwavelength is not required for activation and the extinction coefficientof the Buckminsterfullerene is very high. Moreover, photochemicaleffects should be reduced compared to those produced using an organicdye.

[0046]FIG. 4 is a cross sectional view of an embodiment of the presentinvention wherein a broadband energy-absorbing material 40 is formed ona substrate 42 and a thermoplastic LCM transfer film 44 is affixed tothe surface of the broadband energy-absorbing material. The broadbandenergy-absorbing material 40 is preferably a metal film such as nichromeor titanium. As may be seen from FIG. 4, substrate 42 is in the shape ofa cap for a biological analysis vessel as disclosed in co-pendingapplication Ser. No. ______ filed Feb. 7, 1997, entitled “LASER CAPTUREMICRODISSECTION ANALYSIS VESSEL” expressly incorporated herein byreference. The biological analysis vessel may be a vessel such as aneppindorf tube or other well-known vessel.

[0047]FIG. 5 is a perspective view of a cap 42 for an analysis vesselillustrating a step in a first procedure for affixing the LCM filmthereto. Persons of ordinary skill in the art will observe that cap 42is equipped with a marking means such as a UPC label or laser etchedlabel. Serializing all of the caps provides for easy identification andtracking of cell samples. The label may be read by a sensor, such as aUPC label sensor or OCR sensor, which is mounted in or on the lasercapture microdissection apparatus.

[0048] According to this aspect of the present invention, the serialnumber is placed on the top of the cap. The serial number is placed onthe top of cap 42, and the thickness of cap 42 is selected to be largerthan the depth of field of the microscope objective of the LCM apparatuswith which cap 42 will be used. Thus, the microscope can be focused onthe tissue sample below the bottom surface of the cap and not have thelabel or serial number interfere optically, since the label is far fromthe focal plane of the imaging lens and is thus out of focus.

[0049] There are several ways in which the LCM transfer film 44 may beaffixed to the surface of the caps according to the present invention.As may be seen from FIG. 5, first, a small, e.g., about 1 cm squarepiece of thermoplastic film 44 is cut. The film 44 is gently pressedonto the bottom surface of the cap 42 making it stick thereto. A glassmicroscope slide 46 is heated to about 100 C on a hot plate. A 0.002inch thick piece of mylar plastic release liner 48 is placed on theslide. As is known in the art, a release liner is a plastic sheet thatis coated with a silicone coating so it does not stick to thethermoplastic film material or the glass slide.

[0050] The cap 42 with its attached film 44 is pressed onto the releaseliner/slide assembly for about 5 seconds or until the film melts. Thecap 42 with attached film 44 and release liner 48 is then removed fromthe hot glass slide 46, cooled down to room temperature, and the releaseliner 48 is peeled off. Finally, the excess thermoplastic film istrimmed off.

[0051]FIG. 6 is a perspective view of a cap for an analysis vesselshowing an alternate procedure for affixing the LCM film thereto.According to this method, a piece of transparent double-sided adhesivetape 50 (such as standard double stick tape available from 3MCorporation) may be used to tape the thermoplastic film 44 to the bottomof the cap 42. The excess thermoplastic film may then be trimmed off.

[0052] There are several advantages inherent in the LCM transfer film ofthe present invention. The metal film layer can be very thin and stillhave very high optical absorption. The spin coating process givesoptical quality films of adjustable thickness. In addition, the film ofthe present invention is not sensitive to the wavelength of the incidentlaser light as are the prior art dye impregnated films.

[0053] Another advantage of the present invention is that the broadbandenergy-absorbing material may be isolated from the tissue sample by theEVA transfer film layer. The broadband energy-absorbing material of thepresent invention does not use an organic dye that might havedetrimental photochemistry effects on the sample with which it is incontact. Also, in embodiments of the present invention, which employ ametal film, the metal film does not have to be dissolved into the EVApolymer at high concentrations like an organic dye in the prior artfilms.

[0054] While embodiments and applications of this invention have beenshown and described, it would be apparent to those skilled in the artthat many more modifications than mentioned above are possible withoutdeparting from the inventive concepts herein. The invention, therefore,is not to be restricted except in the spirit of the appended claims.

What is claimed is:
 1. A LCM tissue transfer film comprising athermoplastic film thermally coupled to a broadband energy-absorbingmaterial.
 2. The LCM transfer film of claim 1 wherein said broadbandenergy-absorbing material absorbs from about 10% to about 95% ofincident light.
 3. The LCM tissue transfer film of claim 1 wherein saidbroadband energy-absorbing material is introduced into the filmcomposition as a dopant.
 4. The LCM transfer film of claim 3 whereinsaid broadband energy-absorbing material comprises Buckminsterfullerine.4. The LCM transfer film of claim 3 wherein said broadbandenergy-absorbing material absorbs from about 10% to about 95% ofincident light.
 5. The LCM tissue transfer film of claim 1 wherein saidthermoplastic film is an ethyl vinyl acetate (EVA) film and saidbroadband energy-absorbing material is introduced into the filmcomposition as a dopant.
 6. The LCM tissue transfer film of claim 5wherein said dopant comprises Buckminsterfullerine.
 7. The LCM tissuetransfer film of claim 5 wherein said thermoplastic film has a thicknessof about 100 microns.
 8. The LCM tissue transfer film of claim 1 whereinsaid broadband energy-absorbing material is a layer in thermal contactwith the film.
 9. The LCM tissue transfer film of claim 8 wherein saidbroadband energy-absorbing material is a metal film layer.
 10. The LCMtissue transfer film of claim 9 wherein said broadband energy-absorbingmaterial is a titanium film layer.
 11. The LCM transfer film of claim 10wherein said broadband energy-absorbing material absorbs from about 10%to about 95% of incident light.
 12. The LCM tissue transfer film ofclaim 9 wherein said broadband energy-absorbing material is a nichromefilm layer.
 13. The LCM transfer film of claim 12 wherein said broadbandenergy-absorbing material absorbs from about 10% to about 95% ofincident light.
 14. The LCM tissue transfer film of claim 1 wherein saidthermoplastic film is an ethyl vinyl acetate (EVA) film and saidbroadband energy-absorbing material is a layer in thermal contact withthe film.
 15. The LCM tissue transfer film of claim 14 wherein saidthermoplastic film has a thickness of about 100 microns.
 16. The LCMtissue transfer film of claim 14 wherein said broadband energy-absorbingmaterial is a metal film layer.
 17. The LCM tissue transfer film ofclaim 16 wherein said broadband energy-absorbing material is a titaniumfilm layer.
 18. The LCM transfer film of claim 17 wherein said broadbandenergy-absorbing material absorbs from about 10% to about 95% ofincident light.
 19. The LCM tissue transfer film of claim 16 whereinsaid broadband energy-absorbing material is a nichrome film layer. 20.The LCM transfer film of claim 19 wherein said broadbandenergy-absorbing material absorbs from about 10% to about 95% ofincident light.
 21. A film for LCM tissue transfer comprising: a layerof a transparent support film; a layer of a broadband absorbing film;and a layer of a low temperature thermoplastic; said film having, anoverall thickness chosen to absorb a desired fraction of incident laserlight.
 22. The film of claim 21 wherein: said layer of a transparentsupport film comprises a film chosen from the group including mylar andpolyester; said layer of a broadband absorbing film; comprises a metalfilm chosen from the group including nichrome and titanium, and saidlayer of a low temperature thermoplastic comprises ethyl vinyl alcohol(EVA).
 23. The film of claim 21 wherein said desired fraction ofincident laser light is between about 10% to about 90%.
 24. A film forLCM tissue transfer comprising: a layer of a transparent support film; afirst of a low temperature thermoplastic; layer of a broadband absorbingfilm; and a second layer of a low temperature thermoplastic; saidabsorbing film having an overall thickness chosen to absorb a desiredfraction of incident laser light.
 25. The film of claim 24 wherein: saidlayer of a transparent support film comprises a film chosen from thegroup including mylar and polyester; said layer of a broadband absorbingfilm; comprises a metal film chosen from the group including nichromeand titanium, and said first and second layers of a low temperaturethermoplastic comprise ethyl vinyl alcohol (EVA).
 26. The film of claim24 wherein said desired fraction of incident laser light is betweenabout 10% to about 90%.
 27. A carrier for LCM analysis comprising: asubstrate; and a LCM film mounted on said substrate, said LCM filmcomprising a thermoplastic film thermally coupled to a broadbandenergy-absorbing material.
 28. The carrier of claim 27 wherein saidcarrier is transparent at least in a region where said film is mountedthereon.
 29. The carrier of claim 27 wherein said carrier comprises aplastic material.
 30. The carrier of claim 27 wherein said substrate isadapted to mate with an analysis vessel.
 31. The carrier of claim 27wherein said substrate is adapted to mate with a holder in an LCM tissuesample transfer apparatus.
 32. A cap for sealing a laser capturemicrodissection sample vessel having an open end, said cap comprising: aplastic member adapted to mate with the open end of said sample vesselso as to seal said vessel, said member having a top surface and a bottomsurface and further having an aperture formed from said top surface tosaid bottom surface; and a laser capture microdissection lift-offsubstrate mounted on said bottom surface across and sealing saidaperture.
 33. The cap of claim 32 having a thickness between said topsurface and said bottom surface selected to be larger than the depth offield of a selected microscope objective, said cap further having alabel on said top surface.
 34. A cap for sealing a laser capturemicrodissection sample vessel having an open end, said cap comprising: aplastic member adapted to mate with the open end of said sample vesselso as to seal said vessel, said member having transparent opticalquality windows on opposing top and bottom surfaces defining a solidvolume thereof; and a laser capture microdissection lift-off substrateaffixed to said window on said bottom surface.
 35. The cap of claim 12having a thickness between said top surface and said bottom surfaceselected to be larger than the depth of field of a selected microscopeobjective, said cap further having a label on said top surface.
 36. Amethod for fabricating a film for LCM tissue transfer comprising thesteps of: providing a support layer; forming a layer of a broadbandabsorbing film onto said support layer, said broadband absorbing filmhaving a thickness chosen to absorb a desired fraction of incident laserlight; coating said layer of broadband absorbing film with a layer ofthermoplastic.
 37. A method for fabricating a fihn for LCM tissuetransfer comprising the steps of: providing a polyester support layer;forming a thin layer of metal film onto said polyester support layer,said metal film having a thickness chosen to absorb a desired fractionof incident laser light; coating said metal film with a layer of EVAthermoplastic.
 38. The method of claim 37 wherein said forming stepcomprises a sputtering step and said coating step comprises at least onespin coating step.
 39. The method of claim 38 wherein said coating stepcomprises a plurality of spin coating steps.
 40. A method forfabricating a film for LCM tissue transfer comprising the steps of:providing a support layer; forming a first layer of thermoplastic onsaid support layer; forming a layer of a broadband absorbing film ontosaid first layer of thermoplastic, said broadband absorbing film havinga thickness chosen to absorb a desired fraction of incident laser light;coating said layer of broadband absorbing film with a second layer ofthermoplastic.
 41. A method for fabricating a film for LCM tissuetransfer comprising the steps of: providing a polyester support layer;forming a first layer of EVA thermoplastic on said support layer;forming a thin layer of metal film onto said first layer of EVAthermoplastic, said metal film having a thickness chosen to absorb adesired fraction of incident laser light; coating said metal film with asecond layer of EVA thermoplastic.
 42. The method of claim 41 whereinsaid forming step comprises a sputtering step and said coating stepcomprises at least one spin coating step.
 43. The method of claim 44wherein said coating step comprises a plurality of spin coating steps.